The present invention relates to antitoxin therapy and prevention of disease due to Escherichia coli verotoxin in humans and other animals, and diagnostic assays to detect toxins. In particular, the present invention relates to the isolation of polypeptides derived from Escherichia coli verotoxins, and the use thereof as immunogens for the production of vaccines, including multivalent vaccines, and antitoxins.
A. Escherichia coli as a Pathogenic Organism
Escherichia coli is the organism most commonly isolated in clinical microbiology laboratories, as it is usually present as normal flora in the intestines of humans and other animals. However, it is an important cause of intestinal, as well as extraintestinal infections. For example, in a 1984 survey of nosocomial infections in the United States, E. coli was associated with 30.7% of the urinary tract infections, 11.5% of the surgical wound infections, 6.4% of the lower respiratory tract infections, 10.5% of the primary bacteremia cases, 7.0% of the cutaneous infections, and 7.4% of the other infections (J. J. Farmer and M. T. Kelly, xe2x80x9cEnterobacteriaceae,xe2x80x9d in Manual of Clinical Microbiology, Balows et al.(eds), American Society for Microbiology, [1991], p. 365). Surveillance reports from England, Wales and Ireland for 1986 indicate that E. coli was responsible for 5,473 cases of bacteremia (including blood, bone marrow, spleen and heart specimens); of these, 568 were fatal. For spinal fluid specimens, there were 58 cases, with 10 fatalities (J. J. Farmer and M. T. Kelly, xe2x80x9cEnterobacteriaceae,xe2x80x9d in Manual of Clinical Microbiology, Balows et al.(eds), American Society for Microbiology, [1991], p. 366). There are no similar data for United States, as these are not reportable diseases in this country.
Studies in various countries have identified certain serotypes (based on both the O and H antigens) that are associated with the four major groups of E. coli recognized as enteric pathogens. Table 1 lists common serotypes included within these groups. The first group includes the classical enteropathogenic serotypes (xe2x80x9cEPECxe2x80x9d); the next group includes those that produce heat-labile or heat-stable enterotoxins (xe2x80x9cETECxe2x80x9d); the third group includes the enteroinvasive strains (xe2x80x9cEIECxe2x80x9d) that mimic Shigella strains in their ability to invade and multiply within intestinal epithelial cells; and the fourth group includes strains and serotypes that cause hemorrhagic colitis or produce Shiga-like toxins (or verotoxins) (xe2x80x9cVTECxe2x80x9d or xe2x80x9cEHECxe2x80x9d [enterohemmorrhagic E. coli]).
B. Verotoxin Producing Strains of E. coli 
Although all of these disease-associated serotypes cause potentially life-threatening disease, E. coli O157:H7 and other verotoxin-producing strains have recently gained widespread public attention in the United States due to their recently recognized association with two serious extraintestinal diseases, hemolytic uremic syndrome (xe2x80x9cHUSxe2x80x9d) and thrombotic thrombocytopenic purpura (xe2x80x9cTTPxe2x80x9d). Worldwide, E. coli O157:H7 and other verotoxin-producing E. coli (VTEC) are an increasingly important human health problem. First identified as a cause of human illness in early 1982 following two outbreaks of food-related hemorrhagic colitis in Oregon and Michigan (M. A. Karmali, xe2x80x9cInfection by Verocytotoxin-Producing Escherichia coli,xe2x80x9d Clin. Microbiol. Rev., 2:15-38 [1989]; and L. W. Riley, et al. xe2x80x9cHemorrhagic colitis associated with a rare Escherichia coli serotype,xe2x80x9d New Eng. J. Med., 308: 681-685 [1983]), the reported incidence of VTEC-associated disease has risen steadily, with outbreaks occurring in the U.S., Canada, and Europe.
With increased surveillance, E. coli O157:H7 has been recognized in other areas of the world including Mexico, China, Argentina, Belgium, and Thailand (N. V. Padhye and M. P. Doyle, xe2x80x9cEscherichia coli O157:H7: Epidemiology, pathogenesis and methods for detection in food,xe2x80x9d J. Food. Prot., 55: 555-565 [1992]; and P. M. Griffin and R. V. Tauxe, xe2x80x9cThe epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome,xe2x80x9d Epidemiol. Rev., 13: 60 [1991]).
The disease attracted national attention in the U.S. after a major outbreak in the Pacific Northwest that was associated with consumption of undercooked E. coli O157:H7-contaminated hamburgers. Over 700 hundred people fell ill (more than 170 were hospitalized) and four young children died (P. Recer, xe2x80x9cExperts call for irradiation of meat to protect against food-borne bacteria,xe2x80x9d Associated Press, Jul. 12. 1994 [1994]). Several outbreaks since then have underscored the potential severity and multiple mechanisms for transmission of VTEC-associated diseases (M. Bielaszewska et al., xe2x80x9cVerotoxigenic (enterohaemorrhagic) Escherichia coli in infants and toddlers in Czechoslovakia,xe2x80x9d Infection 18: 352-356 [1990]; A. Caprioli et al., xe2x80x9cHemolytic-uremic syndrome and Vero cytotoxin-producing Escherichia coli infection in Italy, xe2x80x9cJ. Infect. Dis., 166: 184-158 [1992]; A. Caprioli, et al., xe2x80x9cCommunity-wide Outbreak of Hemolytic-Uremic Syndrome Associated with Non-O157 Verocytotoxin-Producing Escherichia coli,xe2x80x9d J. Infect. Dis., 169: 208-211 [1994]; N. Cimolai, xe2x80x9cLow frequency of high level Shiga-like toxin production in enteropathogenic Escherichia coli serogroups,xe2x80x9d Eur. J. Pediatr., 151: 147 [1992]; and R. Voelker., xe2x80x9cPanel calls E. coli screening inadequate,xe2x80x9d Escherichia coli O157:H7xe2x80x94Panel sponsored by the American Gastroenterological Association Foundation in July 1994, Medical News and Perspectives, J. Amer. Med. Assoc., 272: 501 [1994]).
While O157:H7 is currently the predominant E. coli serotype associated with illness in North America, other serotypes (as shown in Table 1, and in particular O26:H11, O113:H21, O91:H21 and O111:NM) also produce verotoxins which appear to be important in the pathogenesis of gastrointestinal manifestations and the hemolytic uremic syndrome (P. M. Griffin and R. V. Tauxe, xe2x80x9cThe epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome,xe2x80x9d Epidemiol. Rev., 13: 60 (1991) M. M. Levine, et al., xe2x80x9cAntibodies to Shiga holotoxin and to two synthetic peptides of the B subunit in sera of patients with Shigella dysenteriae 1 dysentery,xe2x80x9d J. Clin. Microbiol., 30: 1636-1641 [1992]; and C. R. Dorn, et al., xe2x80x9cProperties of Vero cytotoxin producing Escherichia coli of human and animal origin belonging to serotypes other than O157:H7,xe2x80x9d Epidemiol. Infect., 103: 83-95 [1989]). Since organisms with these serotypes have been shown to cause illness in humans they may assume greater public health importance over time (P. M. Griffin and R. V. Tauxe, xe2x80x9cThe epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome,xe2x80x9d Epidemiol. Rev., 13: 60 (1991).
Clinicians usually observe cases of hemolytic uremic syndrome (xe2x80x9cHUSxe2x80x9d) clustered in a geographic region. However, small outbreaks are likely to be missed because many laboratories do not routinely screen stool specimens for E. coli O157:H7. Many cases related to non-commercial food preparation also probably go unrecognized. Nonetheless, E. coli O157:H7 is responsible for a large number of cases, as more than 20,000 cases of E. coli O157:H7 infection are reported annually in the U.S., with 400-500 deaths from HUS. However, these estimates were compiled when only 11 states mandated reporting of E. coli O157:H7. Twenty-nine states have recently made E. coli O157:H7 infection a reportable disease (R. Voelker, xe2x80x9cPanel calls E. coli screening inadequate; Escherichia coli O157:H7; panel sponsored by the American Gastroenterological Association Foundation in July 1994, Medical News and Perspectives,xe2x80x9d J. Amer. Med. Assoc., 272: 501 [1994]). Indeed, the Centers for Disease Control recently added E coli O157:H7 to their list of reportable diseases (xe2x80x9cPublic Health Threats,xe2x80x9d Science 267:1427 [1995]).
C. Nature of Verotoxin-Induced Disease
Risk factors for HUS progression following infection with E. coli O157:H7 include age (very young or elderly), bloody diarrhea, leukocytosis, fever, large amounts of ingested pathogen, previous gastrectomy, and the use of antimicrobial agents (in particular, trimethoprim-sulfamethoxazole)(A. A. Harris et al., xe2x80x9cResults of a screening method used in a 12 month stool survey for Escherichia coli O157:H7,xe2x80x9d J. Infect. Dis., 152: 775-777 [1985]; and M. A. Karmali, xe2x80x9cInfection by Verocytotoxin-producing Escherichia coli,xe2x80x9d Clin. Microbiol. Rev., 2: 15-38 [1989]).
As indicated above, E. coli O157:H7 is associated with significant morbidity and mortality. The spectrum of illness associated with E. coli O157:H7 infection includes asymptomatic infection, mild uncomplicated diarrhea, hemorrhagic colitis, HUS, and TTPxe2x80x9d. Hemorrhagic colitis (or xe2x80x9cischemic colitisxe2x80x9d) is a distinct clinical syndrome characterized by sudden onset of abdominal crampsxe2x80x94likened to the pain associated with labor or appendicitisxe2x80x94followed within 24 hours by watery diarrhea. One to two days later, the diarrhea turns grossly bloody in approximately 90% of patients and has been described as xe2x80x9call blood and no stoolxe2x80x9d (C. H. Pai et al., xe2x80x9cSporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7,xe2x80x9d Ann. Intern. Med., 101: 738-742 [1984]; and R. S. Remis et al., xe2x80x9cSporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7,xe2x80x9d Ann. Intern. Med., 101: 738-742 [1984]). Vomiting may occur, but there is little or no fever. The time from ingestion to first loose stool ranges from 3-9 days (with a mean of 4 days) L. W. Riley et al., xe2x80x9cHemorrhagic colitis associated with a rare Escherichia coli serotype,xe2x80x9d New Eng. J. Med., 308: 681-685 [1983]; and D. Pudden et al., xe2x80x9cHemorrhagic colitis in a nursing home,xe2x80x9d Ontario Can. Dis. Weekly Rpt., 11: 169-170 [1985]), and the duration of illness ranges generally from 2-9 days (with a mean of 4 days).
HUS is a life-threatening blood disorder that appears within 3-7 days following onset of diarrhea in 10-15% of patients. Those younger than 10 years and the elderly are at particular risk. Symptoms include renal glomerular damage, hemolytic anemia (rupturing of erythrocytes as they pass through damaged renal glomeruli), thrombocytopenia and acute kidney failure. Approximately 15% of patients with HUS die or suffer chronic renal failure. Indeed, HUS is a leading cause of renal failure in childhood (reviewed by M. A. Karmali, xe2x80x9cInfection by Verocytotoxin-producing Escherichia coli,xe2x80x9d Clin. Microbiol. Rev., 2: 15-38 [1989]). Currently, blood transfusion and dialysis are the only therapies for HUS.
TTP shares similar histopathologic findings with HUS, but usually results in multiorgan microvascular thrombosis. Neurological signs and fever are more prominent in TTP, compared with HUS. Generally occurring in adults, TTP is characterized by microangiopathic hemolytic anemia, profound thrombocytopenia, fluctuating neurologic signs, fever and mild azotemia (H. C. Kwaan, xe2x80x9cClinicopathological features of thrombotic thrombocytopenic purpura,xe2x80x9d Semin. Hematol., 24: 71-81 [1987]; and S. J. Machin, xe2x80x9cClinical annotation: Thrombotic thrombocytopenic purpura,xe2x80x9d Br. J. Hematol., 56: 191-197 [1984]). Patients often die from microthrombi in the brain. In one review of 271 cases, a rapidly progressive course was noted, with 75% of patients dying within 90 days (E. L. Amorosi and J. E. Ultmann, xe2x80x9cThrombotic thrombocytopenic purpura: Report of 16 cases and review of the literature,xe2x80x9d Med., 45:139-159 (1966).
Other diseases associated with E. coli O157:H7 infection include hemorrhagic cystitis and balantitis (W. R. Grandsen et al., xe2x80x9cHemorrhagic cystitis and balantitis associated with verotoxin-producing Escherichia coli O157:H7,xe2x80x9d Lancet ii: 150 [1985]), convulsions, sepsis with other organisms and anemia (P. C. Rowe et al., xe2x80x9cHemolytic anemia after childhood Escherichia coli O157:H7 infection: Are females at increased risk?xe2x80x9d Epidemiol. Infect., 106: 523-530 [1991]).
D. Mechanism of Pathogenesis
Verotoxins are strongly linked to E. coli O157:H7 pathogenesis. All clinical isolates of E. coli O157:H7 have been shown to produce one or both verotoxins (VT1 and VT2) (C. A. Bopp et al., xe2x80x9cUnusual Verotoxin-producing Escherichia coli associated with hemorrhagic colitis,xe2x80x9d J. Clin. Microbiol., 25: 1486-1489 [1987]). The VT1 and VT2 genes are carried by temperate coliphages 933J and 933W, respectively. Once lysogenized, these coliphages lead to the expression of toxin genes by the E. coli host.
Both of these toxins are cytotoxic to Vero (African green monkey kidney) and HeLa cells, and cause paralysis and death in mice (A. D. O""Brien et al., xe2x80x9cPurification of Shigella dysenteriae 1 (Shiga) like toxin from Escherichia coli O157:H7 strain associated with hemorrhagic colitis,xe2x80x9d Lancet ii: 573 [1983]). These toxins are sometimes referred to in the literature as Shiga-like toxins I and II (SLT-I and SLT-II, respectively), due to their similarities with the toxins produced by Shigella. Indeed, much of our understanding of E. coli VTs is based on information accumulated on Shiga toxins. Shiga toxin, first described in 1903, has been recognized as one of the most potent bacterial toxins for eukaryotic cells (reviewed by M. A. Karmali, xe2x80x9cInfection by Verocytotoxin-producing Escherichia coli,xe2x80x9d Clin. Microbiol. Rev., 2: 15-38 [1989]). Hereinafter, the VT convention will be used; thus, VT1 and VT2 correspond to SLT-I and SLT-II, respectively.
While the pathogenic mechanism of E. coli O157:H7 infection is incompletely understood, it is believed that ingested organisms adhere to and colonize the intestinal mucosa, where toxins are released which cause endothelial cell damage and bloody diarrhea. It is also postulated that hemorrhagic colitis progresses to HUS when verotoxins enter the bloodstream, damaging the endothelial cells of the microvasculature and triggering a cascade of events resulting in thrombus deposition in small vessels. These microthrombi occlude the microcapillaries of the kidneys (particularly in the glomeruli) and other organs, resulting in their failure (J. J. Byrnes and J. L. Moake, xe2x80x9cTTP and HUS syndrome: Evolving concepts of pathogenesis and therapy,xe2x80x9d Clin. Hematol., 15: 413-442 [1986]; and T. G. Cleary, xe2x80x9cCytotoxin-producing Escherichia coli and the hemolytic uremic syndrome,xe2x80x9d Pediatr. Clin. North Am., 35: 485-501 [1988]). Verotoxins entering the bloodstream may also result in direct kidney cytotoxicity.
VT1 is immunologically and structurally indistinguishable from Shiga toxin produced by Shigella dysenteriae (A. D. O""Brien et al., xe2x80x9cPurification of Shigella dysenteriae 1 (Shiga) like toxin from Escherichia coli O157:H7 strain associated with hemorrhagic colitis,xe2x80x9d Lancet ii: 573 [1983]). VT1 and VT2 holotoxins each consist of one A and five B subunits (A. Donohue-Rolfe et al., xe2x80x9cPurification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinty chromatography with immobilized P1 glycoprotein and production of cross reactive monoclonal antibodies,xe2x80x9d Infect. Immun., 57: 3888-3893 [1989]; and A. Donohue-Rolfe et al., xe2x80x9cSimplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies,xe2x80x9d J. Exp. Med., 160: 1767-1781 [1984]). Intra-chain disulfide bonds are formed and the holotoxin is assembled after secretion of the subunits to the periplasm. Each subunit contains a leader sequence that targets secretion of the toxin. VT1 and VT2 are structurally related, sharing 56% amino acid homology.
The toxic A subunit is enzymatically active, while the B subunit binds the holotoxin to the receptor on the target eukaryotic cell. The A chain is structurally related to the ricin A chain, and acts in a similar manner to inhibit protein synthesis by cleaving a single adenine residue from 28S ribosomal RNA (Endo et al., J. Biol. Chem., 262:5908-5912 [1987]). The A chain is 32 (VT1) or 33 (VT2) kd in size, and is proteolytically cleaved into A1 (approximately 27 kd) and A2 (approximately 3-4 kd) fragments. In both VT1 and VT2, the non-toxic B subunit is approximately 8 kd. Pentamers of the B subunit bind mammalian cell surface receptors, facilitating internalization of holotoxin by cells.
Crystal structure analysis of Shiga holotoxin and VT1B subunit pentamers have shown that the holotoxin assembles with the C-terminal end of the A subunit associating with, and inserting within, a pentamer of B chains (P. E. Stein et al., xe2x80x9cCrystal structure of the cell-binding B oligomer of vertoxin-1 from E. coli,xe2x80x9d Nature 355: 748-750 [1992]; and M. E. Fraser et al., xe2x80x9cCrystal structure of the holotoxin from Shigella dysenteriae at 2.5 xc3x85 resolution,xe2x80x9d Struct. Biol., 1:59-64 [1994]). The alpha helical C-terminal region of the A chain (residues 279-293) is encircled by a pentameric ring of B subunits, with the remainder of the A chain exposed. This conformation is consistent with the observation that a C-terminally truncated A1 subunit of VT1 is toxic (in a ribosomal inhibition assay), but cannot associate with B subunit pentamers (P. R. Austin et al, xe2x80x9cEvidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity,xe2x80x9d Infect. Immun., 62: 1768 [1994]).
The Verotoxin A Subunit. Examination of the crystal structure of Shiga holotoxin indicates that the N-terminus of its A subunit is both surface-exposed and functionally important. Removal of amino acid interval 3-18 of the A subunit completely abolished toxicity (L. P. Perera et al., xe2x80x9cMapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II,xe2x80x9d Infect. Immun., 59: 829-835 [1991]) while removal of interval 25-44 retained toxicity but abolished its association with B subunit pentamers (J. E. Haddad et al., xe2x80x9cMinimum domain of the Shiga toxin A subunit required for enzymatic activity,xe2x80x9d J. Bacteriol., 175: 4970-4978 [1993]). Deletion of the first 13 residues of the homologous ricin A subunit also abolished toxicity, while deletion of the first 9 residues did not (M. J. May, et al., xe2x80x9cRibosome inactivation by ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides,xe2x80x9d EMBO J., 8: 301-308 [1989]).
The Verotoxin B Subunit. Studies of Shiga toxin B subunit suggest that neutralizing epitopes may also be present at both the N- and C-terminal regions of VT1 and VT2 B subunits. Polyclonal antibodies raised against peptides from these regions (residues 5-18, 13-26, 7-26, 54-67 and 57-67) show partial neutralization of Shiga toxin (I. Harari and R. Arnon, xe2x80x9cCarboxy-terminal peptides from the B subunit of Shiga toxin induce a local and parenteral protective effect,xe2x80x9d Mol. Immunol., 27: 613-621 [1990]; and I. Harari et al., xe2x80x9cSynthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity,xe2x80x9d Infect. Immun., 56: 1618-1624 [1988]). Deletion of the last five amino acids of Shiga toxin B (M. P. Jackson et al., xe2x80x9cFunctional Analysis of the Shiga toxin and Shiga-like toxin Type II variant binding subunits by using site-directed mutagenesis,xe2x80x9d J. Bacteriol., 172: 653-658 [1990]), or four amino acids of VT2 B (L. P. Perera et al., xe2x80x9cMapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II,xe2x80x9d Infect. Immun., 59: 829-835 [1991]), eliminate toxin activity, while deletion of the last two amino acids of VT2 B subunit reduced cytotoxicity. In contrast, the addition of an 18 or 21 amino acid extension to the native C-terminus of the VT2 B subunit was presumably conformationally correct, as these proteins assembled cytotoxic holotoxin.
Various approaches to express recombinant verotoxins have included individual or coordinate expression of A and B subunits from high-copy number plasmids and expression with fusion partners (J. E. Haddad et al., xe2x80x9cMinimum domain of the Shiga toxin A subunit required for enzymatic activity,xe2x80x9d J. Bacteriol., 175: 4970-4978; J. E. Haddad, and M. P. Jackson, xe2x80x9cIdentification of the Shiga toxin A-subunit residues required for holotoxin assembly,xe2x80x9d J. Bacteriol., 175: 7652-7657 [1993]; M. P. Jackson et al., xe2x80x9cMutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits,xe2x80x9d J. Bacteriol., 172: 3346-3350 [1990]; C. J. Hovde et al., xe2x80x9cEvidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I,xe2x80x9d Proc. Natl. Acad. Sci., 85: 2568-2572 [1988]; R. L. Deresiewicz et al., xe2x80x9cThe role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain,xe2x80x9d Mol. Gen. Genet., 241: 467-473 [1993]; T. M. Zollman et al., xe2x80x9cPurification of Recombinant Shiga-like Toxin Type I A1 Fragment from Escherichia coli,xe2x80x9d Protein Express. Purific., 5: 291-295 [1994]; K. Ramotar, et al., xe2x80x9cCharacterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron,xe2x80x9d Biochem J., 272: 805-811 [1990]; S. B. Calderwood et al., xe2x80x9cA system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit,xe2x80x9d Infect. Immun., 58: 2977-2982 [1990]; D. W. K. Acheson, et al., xe2x80x9cComparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems,xe2x80x9d Infect. Immun. 61: 1098-1104 [1993]; M. P. Jackson et al., xe2x80x9cNucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933,xe2x80x9d FEMS Microbiol. Lett., 44: 109-114 [1987]; J. W. Newland et al., xe2x80x9cCloning of genes for production of Escherichia coli Shiga-like toxin type II,xe2x80x9d Infect. Immun. 55: 2675-2680 [1987]; and F. Gunzer and H. Karch, xe2x80x9cExpression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology,xe2x80x9d J. Clin. Microbiol., 31: 2604-2610 [1993]; and D. W. Acheson et al., xe2x80x9cExpression and purification of Shiga-like toxin II B subunits,xe2x80x9d Infect. Immun., 63:301-308 [1995]). In one case, bench top fermentation techniques yielded 22 mg/liter of soluble recombinant protein (D. W. K. Acheson, et al., xe2x80x9cComparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or Iron-regulated promoter systems,xe2x80x9d Infect. Immun. 61: 1098-1104 [1993]). However, there have been no systematic approaches to identifying the appropriate spectrum of VT antigens, preserving immunogen and immunoabsorbant antigenicity and scaling-up.
The receptor for VT1 and VT2 is a globotriaosyl ceramide containing a galactose xcex1-(1-4)-galactose-xcex2-(1-4) glucose ceramide (Gb3) (C. A. Lingwood et al., xe2x80x9cGlycolipid binding of natural and recombinant Escherichia coli produced verotoxin in vitro,xe2x80x9d J. Biol. Chem., 262: 1779-1785 [1987]; and T. Wadell et al., xe2x80x9cGlobotriaosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2,xe2x80x9d Biochem. Biophys. Res. Commun., 152: 674-679 [1987]). Gb3 is abundant in the cortex of the human kidney and is present in primary human endothelial cell cultures. Hence, the identification of Gb3 as the functional receptor for VT1 and VT2 is consistent with their role in HUS pathogenesis, in which endothelial cells of the renal vasculature are the principal site of damage. Therefore, toxin-mediated pathogenesis may follow a sequence of B subunit binding to Gb3 receptors on kidney cells, toxin internalization, enzymatic reduction of the A subunit to an A1 fragment, binding of the A1 subunit to the 60S ribosomal subunit, inhibition of protein synthesis and cell death (A. D. O""Brien et al., xe2x80x9cShiga and Shiga-like toxins. Microbial Rev., 51: 206-220 [1987]).
The role of verotoxins in the pathogenesis of E. coli O157:H7 infections has been further studied in animal models. Infection or toxin challenge of laboratory animals do not produce all the pathologies and symptoms of hemorrhagic colitis, HUS, and TTP which occur in humans. Glomerular damage is noticeably absent. Nonetheless, experiments using animal models implicate verotoxins as the direct cause of hemorrhagic colitis, microvascular damage leading to the failure of kidneys and other organs and CNS neuropathies.
For example, Barrett, et al. delivered VT2 into the peritoneal cavity of rabbits using mini-osmotic pumps (J. J. Barrett et al., xe2x80x9cContinuous peritoneal infusion of shiga-like toxin II (SLTII) as a model for SLT II-induced diseases,xe2x80x9d J. Infect. Dis., 159: 774-777 [1989]). In three days, most animals receiving the toxin developed diarrhea, with intestinal lesions resembling those seen in humans with hemorrhagic colitis. Although there was some evidence of renal dysfunction, none of the rabbits developed HUS. Beery, et al. showed that VT2, when administered intraperitoneally or intravenously to adult mice, produces lesions of the kidneys and colon (J. T. Beery et al., xe2x80x9cCytotoxic activity of Escherichia coli O157:H7 culture filtrate on the mouse colon and kidney,xe2x80x9d Curr. Microbiol., 11: 335-342 [1984]). Histologic lesions in the kidney included accumulation of numerous exfoliated collecting tubules and marked intracellular vacuolation of proximal convoluted tubular cells.
Sjxc3x6gren et. al. studied the pathogenesis of an entero-adherent strain of E. coli (RDEC-1) lysogenized with a VT1-containing bacteriophage (VT1-producing RDEC-1) (R. Sjxc3x6gren et al., xe2x80x9cRole of Shiga-like toxin I in bacterial enteritis: comparison between isogenic Escherichia coli strains induced in rabbits,xe2x80x9d Gastroenterol., 106: 306-317 [1994]). In this study, rabbits were challenged with RDEC-1 or VT1-producing RDEC-1 and studied for onset of disease. The VT1-producing variant induced a severe, non-invasive, entero-adherent infection in rabbits which was characterized by serious histological lesions with vascular changes, edema and severe epithelial inflammation. Importantly, vascular changes consistent with endothelial damage were seen in infected animals that was similar to intestinal microvascular changes in humans with E. coli O157:H7 infection. Based on these observations, they concluded that VT1 is an important virulence factor in enterohemorrhagic E. coli O157:H7 infection.
Fuji et. al. described a model in which mice were treated for three days with streptomycin followed by a simultaneous challenge of E. coli O157:H7 orally, and mitomycin intraperitoneally (J. Fuji et al., xe2x80x9cDirect evidence of neuron impairment by oral infection with Verotoxin-producing Escherichia coli O157:H7 in mitomycin-treated mice,xe2x80x9d Infect. Immun., 62: 3447-34453 [1994]). All of the animals died within four days. Immunoelectron-microscopy strongly suggested that death was due to the toxic effects of VT2v (a structural variant of VT2), on both the endothelial cells and neurons in the central nervous system which resulted in fatal acute encephalopathy.
Wadolkowski et al. studied colonization of E. coli O157:H7 in mice. Mice were treated with streptomycin and fed 1010 E. coli O157:H7 (E. A. Wadolkowski et al., xe2x80x9cMouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7,xe2x80x9d Infect. Immun., 58: 2438-2445 [1990]; and E. A. Wadolkowski et al., xe2x80x9cAcute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin Type II,xe2x80x9d Infect. Immun., 58: 3959-3965 [1990]). All of the mice died due to severe, disseminated, acute necrosis of proximal convoluted tubules. In mouse models, glomerular damage was not observed, but toxic acute renal tubular necrosis was observed which is characteristic of some HUS patients. The failure of mice to show glomerular damage is thought to be due to the absence of a functional globotriaosyl ceramide receptor specific for verotoxins in the glomeruli of the kidneys. Administration of VT2 subunit-specific monoclonal antibodies prior to infection prevented all pathology and death.
E. Current Therapeutic Approaches
E. coli O157:H7 disease is not adequately controlled by current therapy. Patient treatment is tailored to manage fluid and electrolyte disturbances, anemia, renal failure and hypertension. Although E. coli O157:H7 is susceptible to common antibiotics, the role of antibiotics in the treatment of infection has questionable merit. In both retrospective and prospective studies, prophylaxis or treatment with antibiotics such as trimethoprim-sulfamethoxazole, there was either no benefit or an increased risk of developing HUS (T. N. Bokete et al., xe2x80x9cShiga-like toxin producing Escherichia coli in Seattle children: a prospective study,xe2x80x9d Gastroenterol., 105: 1724-1731 [1993]; A. T. Pavia et al., xe2x80x9cHemolytic uremic-syndrome during an outbreak of Escherichia coli O157:H7 infections in institutions for mentally retarded persons: clinical and epidemiologic observations,xe2x80x9d J. Pedatr., 116: 544-551 [1990]; F. Proulx et al., xe2x80x9cRandomized, controlled trial of antibiotic therapy for Escherichia coli O157:H7 enteritis,xe2x80x9d J. Pediatr. 121: 299-303 [1992]; and A. L. Carter et al., xe2x80x9cA severe outbreak of Escherichia coli O157:H7-associated hemorrhagic colitis in a nursing home,xe2x80x9d New Eng. J. Med., 317: 1496-1500 [1987]).
The mechanisms by which antibiotics increase the risk of infection or related complications might involve enhancement of toxin production, release of toxins from killed organisms, or alteration of normal competing intestinal flora allowing for pathogen overgrowth (M. A. Karmali, xe2x80x9cInfection by Verocytotoxin-producing Escherichia coli,xe2x80x9d Clin. Microbiol. Rev., 2: 15-38 [1989]). A further concern in the use of antibiotics is the potential acquisition of antimicrobial resistance by E. coli O157:H7 (C. R. Dorn, xe2x80x9cReview of foodborne outbreak of Escherichia coli O157:H7 infection in the western United States,xe2x80x9d JAVMA 203: 1583-1587 [1993]).
In addition, by the time symptoms are serious enough to attract medical attention, it is likely that verotoxins are already entering the systemic circulation or will do so shortly thereafter. Although antimicrobials may help to prevent pathology resulting from the action of toxin on the bowel lumen. However, by the time symptoms of HUS have developed, the patient has ceased shedding organisms. Thus, antimicrobial treatment during HUS disease is of less value, and often contraindicated, due to the increased risk of complications associated with administration of antimicrobials to patients susceptible to development of HUS. Importantly, there is currently no antitoxin commercially available for use in treating affected patients. What is needed is a means to block the progression of disease, without the complications associated with antimicrobial treatment.